Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors

A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton. A subset of these samples with DNA availability was tested by ddPCR and UltraSEEK MassARRAY for mutation detection in 5 genes (IDH1, PIK3CA, KRAS, BRAF, and NRAS). Sixty one of 104 expected tissue mutations and 6 additional mutations not present in the tissue were detected in cfDNA. ddPCR and MassARRAY showed 83% and 77% concordance with NGS for mutation detection with 100% and 79% sensitivity, respectively. The median OS of patients with lower cfDNA yield (74 vs 50 months; P < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; p < 0.04) was significantly longer than in patients with higher cfDNA yield and positive for mutations. A limit-of-detection of 0.1% was demonstrated for ddPCR and MassARRAY platforms using a serially diluted positive cfDNA sample. The MassARRAY and ddPCR systems enable fast and cost-effective genotyping for a targeted set of mutations and can be used for single gene testing to guide response to chemotherapy or for orthogonal validation of NGS results.